This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. critically important for therapeutic efficacy. Unfortunately, many methods for drug delivery are often highly inefficient due to non-ideal serum-stability, transport across biological barriers, and release at the target site. To address these problems, we will create bio-responsive, polymeric nano-containers for the selective delivery of therapeutic compounds to pre-determined cellular locations. We will combine the solution selfassembly of block copolymers with selective targeting and cleaving peptide linkages to permit vesicle evolution in response to critical environmental stimuli, eventually leading to site-specific release of the encapsulated payload. In the first specific aim, we will create novel peptide-containing block copolymers and characterize their self-assembly in aqueous solution. These amphiphilic copolymers will be designed to assemble into vesicular structures. Peptides designed to promote endocytotic uptake (1) and endosomal release (2) will be sequentially incorporated into the hydrophilic backbone of the block copolymer in a layered fashion. In the second specific aim, we will evaluate the protease-sensitivity and targeting efficiency of each peptide layer. The protease accessibility of each peptide sequence, as well as vesicle stability, will be assessed as a function of peptide position in the vesicle's corona. The cell-vesicle binding, vesicle internalization, and endosomolytic activity of the vesicles also will be evaluated. In the third specific aim, we will validate the ability of the peptides to direct vesicle transport to and rupture within the cytosol, and we will evaluate the cytotoxicity of both payload-free and cytotoxinincorporating vesicles. Vesicle rupture will be induced by selective placement of peptide (2) proximal to the hydrophobic block of the polymer. Our long-term vision for this project is the development of a nucleic acid delivery system for the selective targeting of tumor stromal fibroblasts and/or inflammatory cells. We envision the complete de novo design of modular vesicular nano-capsules containing a series of location specific "sheddable" shells to direct payload transport in response to environmental cues at each transport barrier.